Type 1 allergies have worldwide importance. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma. These allergies are caused by allergens present in the air (aeroallergens) which are liberated by sources of various origin, such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE reactivity with grass pollen allergens (Freidhoff et al., 1986, J. Allergy Clin. Immunol. 78: 1190-2002). The substances which trigger type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bound to the surface of mast cells in sensitised persons. Crosslinking of two IgE molecules with one another by an allergen results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding clinical symptoms.
Depending on the relative frequency with which the individual allergen molecules react with the IgE antibodies of allergy sufferers, a distinction is made between major and minor allergens.
In the case of timothy grass (Phleum pratense), Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthiesen and Löwenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al., 1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54). Phl p 2/3 (Dolecek et al., 1993, FEBS 335 (3), 299-304), Phl p 4 (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int. Arch. Allergy Immunol. 97: 287-294, Fischer et al., 1996, J. Allergy Clin. Immunol. 98: 189-198) and Phl p 13 (Suck et al., 2000, Clin. Exp. Allergy 30: 324-332; Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402) have hitherto been identified as major allergens.
The dominant major allergens of timothy grass (Phleum pratense) are Phl p 1 and Phl p 5. Since the major allergens of the grasses from the Poaceae family are highly homologous with one another and consequently have very similar biochemical and immunological properties, these related proteins are grouped together as group 1 and group 5 allergens.
The group 1 allergens react with the IgE antibodies in more than 95% of grass pollen allergy sufferers and are thus the dominant major allergens of grass pollen.
The group 1 allergens are glycoproteins having a molecular weight of about 32 kDa and are localised in the cytoplasm of the pollen grains. Both contact of the pollen grains with the mucous membrane of the upper respiratory tract and also moistening of the pollen grains by rain lead to rapid release of these allergens. The rapid release of the group 1 allergens, also in the form of sub-cellular microparticles, enables penetration into the lower respiratory tract, which can result in triggering of severe asthma attacks.
The cDNAs of group 1 allergens from Phleum pretense (Laffer et al., 1994, J. Allergy Clin. Immunol. 94: 689-698), Lolium perenne (Perez et al., 1990, J. Biol. Chem. 265: 16210-16250), Holcus lanatus (Schramm et al., 1997, J. Allergy Clin. Immunol. 1999:781-787), Poa pratensis (Sturaro u. Viotti, 1998, NCBI GenBank, Acc. No. AJ 131850), Cynodon dactylon (Smith et al., 1996, J. Allergy Clin. Immunol. 98: 331-343), Phalaris aquatica (Suphioglu et al., 1995, Clin. Exp. Allergy 25: 853-865) and Oryza sativa (Xu et al., 1995, Gene 164: 255-259) have been identified.
In addition to these first descriptions of the sequence, further group 1 allergen sequences which differ from the original sequences in individual positions have been published in databases. Such isoforms are also known for other grass pollen allergens.
Owing to their homology, the group 1 allergens of the sweet grasses (Poaceae) have high cross-reactivity with human IgE antibodies (Laffer et al., 1996, Mol. Immunology 33: 417-426). This immunological cross-reactivity is based on a very similar amino acid sequence, as shown by a sequence comparison of Phl p 1, the group 1 allergen of timothy grass (Phleum pretense), with group 1 molecules from selected species in FIG. 1.
Homologous sequence regions in the other group 1 allergens of the Poaceae exist both for the sequence regions of the Phl p 1 amino acid sequence deletions described here in the construction of hypoallergenic variants and also for the flanking sequence regions thereof. Furthermore, both the number and also the surrounding sequence regions of the cysteines of the group 1 allergens of the Poaceae are preserved. Owing to sequence homologies, the group 1 allergens of the Poaceae are classified in the protein family of the β-expansins (Cosgrove D. J., 2000 Nature 407: 321-6).
A classical approach to effective therapeutic treatment of allergies is specific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4 (6): 336-339: Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562), where natural allergen extracts are injected subcutaneously into the patient in increasing doses. However, there is a risk in this method of allergic reactions or even anaphylactic shock. In order to minimise these risks, innovative preparations in the form of allergoids are employed. These are chemically modified allergen extracts which have significantly reduced IgE reactivity, but identical T-cell reactivity compared with the untreated extract. These T-cell epitopes are of crucial importance for the therapeutic action of the allergen preparations in hyposensitisation (Fiebig, 1995, Allergo J. 4 (7): 377-382).
A greater degree of therapy optimisation would be possible with allergens prepared by recombinant methods. Defined cocktails of high-purity allergens prepared by recombinant methods, if desired matched to the individual sensitisation patterns of patients, could supersede extracts from natural allergen sources since the latter, in addition to the various allergens, contain a relatively large number of immunogenic, but non-allergenic accompanying proteins.
Realistic perspectives which may result in safe hyposensitisation with recombinant expression products are offered by specifically mutated recombinant allergens in which IgE epitopes are specifically deleted without impairing the T-cell epitopes which are essential for the therapy (Schramm et al., 1999, J. Immunol. 162: 2406-2414).
A different concept for hyposensitisation is based on the fact that a protective immune response is induced, in particular, by IgG4 antibodies with a blocking action. In accordance with this hypothesis, recombinant Phl p 1 fragments have been described which are said to be suitable for induction of a protective IgG4 response (Ball et al., 1999, FASEB J. 13:1277-1290).
This concept is completely different from the concept of hypoallergenic allergen variants having reduced IgE reactivity and maintained T-cell reactivity.
Another possibility for influencing the disturbed T helper cell balance in allergy sufferers by therapeutic methods is treatment with expressible DNA which encodes for the relevant allergens (immunotherapeutic DNA vaccination). Experimental confirmation of the allergen-specific effect on the immune response has been obtained in rodents by injection of allergen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5): 540-544).
The object on which the present invention is based consisted in the provision of novel variants of the group 1 allergens of the Poaceae at the protein and DNA level which are distinguished by reduced IgE activity with substantial maintenance of the T-cell reactivity and are therefore suitable for curative and preventive specific immunotherapy and immunotherapeutic DNA vaccination.
The work which led to the variants found was carried out using Phl p 1 as model allergen. It becomes clear from the sequence alignments shown in FIG. 1 that, owing to the high homology within group 1, the same results would have been obtained if the starting point had been another group 1 allergen.
Thus, it must also be assumed that the results given above and below can also be applied to Sec c 1 from Secale cereale or would have been obtained using Sec c 1, although the sequence is still unknown for this group 1 allergen.
The present invention therefore relates to variants of the group 1 allergens of the Poaceae which are characterised by reduced IgE reactivity compared with the known wild-type allergens and by maintained reactivity with T-lymphocytes. These group 1 allergens are preferably Phl p 1, Poa p 1, Hol p 1, Lol p 1, Cyn d 1, Ory s 1 and Pha a 1 from Phleum pretense, Lolium perenne, Poa pratensis, Holcus lanatus, Cynodon dactylon, Oryza saliva and Phalaris aquatica. Greater preference is given to Phl p 1, Poa p 1, Hol p 1, Lol p 1 or Pha a 1 and very particular preference is given to Phl p 1.
The starting point for the construction of the hypoallergenic variants of the group 1 allergens is the cDNA of wild-type Phl p 1, which was isolated with the aid of specific primers by polymerase chain reaction (PC R) from the total cDNA from pollen of Phleum pretense (“GenBank” entry Z27090; NCBI, Bethesda, USA) (SEQ ID NO 1).
The amino acid sequence SEQ ID NO 2 was deduced from the cDNA sequence of wild-type Phl p 1.
Phl p 1, which consists of 240 amino acids and is glycosylated in the natural form, is—like all group 1 allergens (see FIG. 1)—characterised by the existence of seven cysteines in the mature molecule. With the exception of Cyn d 1 and Ory s 1, these amino acid positions have the numbers 41, 57, 69, 72, 77, 83 and 139 in all group 1 allergens (Petersen et al., 1995, J. Allergy Clin. Immunol 95: 987-994).
Phl p 1 has been expressed in E. coli as non-glycosylated protein. The recombinant wild-type protein (rPhl p 1 wt) react with IgE antibodies from grass pollen allergy sufferers which have reactivity with natural purified Phl p 1 (nPhl p 1) (Petersen et al., 1998, Clin. Exp. Allergy 28: 315-321).